Assay for Chagas&#39; Disease and reagents for its use

ABSTRACT

An assay to confirm the presence of antibodies to T. cruzi in a test sample. The assay comprises detecting the presence of antibody to three T. cruzi antigens, Gp90, Gp 60/50 and LPPG in a test sample. The presence of antibody in the test sample to at least two of three T. cruzi antibodies is indicative of a confirmed reactive sample. Also provided are diagnostic reagents for detection of T. cruzi, a process for purifying GP 60/50, a process for linking a protein and LPPG, and diagnostic test kits for use when assaying for antibodies to T. cruzi.

This is a division of U.S. patent application Ser. No. 08/275,086 filedJul. 13, 1994, now U.S. Pat. No. 5,550,027, which is a continuation ofU.S. patent application Ser. No. 07/911,590 filed Jul. 10, 1992, nowabandoned.

BACKGROUND OF THE INVENTION

This invention relates generally to the detection of parenterallytransmitted diseases and their detection, and more particularly, relatesto Trypanosoma cruzi which is the causative agent of Chagas' Disease,and assays for its detection in test samples.

The protozoan parasite Trypanosoma cruzi is the causative agentresponsible for a disease known as Chagas' Disease or as AmericanTrypanosomiasis. The geographical range of this disease in the Americasis as far north as California and Maryland and as far south as regionsin Argentina and Chile. It has been estimated that 90 million people areat risk, and that an additional 12 to 63 million individuals areinfected with this parasite. See, for example, G. A. Schmunis,Transfusion 31:547-557 (1991); Anonymous, WHO Technical Report Series1991 811:1-93 (1991); and S. Kingman, New Scientist 132:16-17 (1991).The first indigenous case of Chagas' Disease in North America wasreported in 1955. N. C. Woody et al., JAMA 159:676-677 (1955); R. J.Schiffler et al., JAMA 251:2983-84 (1984); J. D. Pearlman, Am. J. Med.75:1057-1060 (1983); T. R. Navin, Am. J. Public Health 75:366-369(1985); and Anonymous, Texas Health Bull. 159:11-13 (1955).

Chagas' Disease is transmissable through blood products; thetransmission of Chagas' Disease through blood transfusion in the UnitedStates has been recognized for some time. Two recent serological surveysin the Washington, D.C. area have indicated that several individualsresiding in that area, originally from El Salvador and Nicaragua, hadsera reactive for Chagas' Disease. L. V. Kirchhoff et al., JAMA254:3058-3060 (1985); and L. V. Kirchhoff et al., Am. J. Med. 82:915-920(1987). It has been estimated from this survey that there are up to100,000 individuals living in the United States who are chronicallyinfected with T. cruzi. A. Skolnick, JAMA 265:173 (1991). In a separatereport, of 1027 consecutive blood donations in Los Angeles County duringa three-month period which were screened by a complement fixation testfor Chagas' Disease, there were ten initial reactives and one confirmedcase. P. Kerndt et al., Transfusion 28:31S Abstract s108 (1988) and P.Kerndt et al., Transfusion 31:814-818 (1991). Imported Chagas' Diseasehas been recognized in Europe as well, with a documented case ofcongenital T. cruzi infection reported in Sweden. P. O. Pehrson et al,Scand. J. Infect. Dis. 13:307-308 (1981).

Trypanosoma cruzi has one of the most complex life cycles oftrypanosomes found in man. Trypomastigotes circulate in the blood ofvertebrate hosts and are transmitted by blood-sucking triatomid insects.The disease also can be spread by blood transfusion, through intravenousdrug use, by congenital transmission, by sexual activity, by organtransplant or through breast milk. See, for example, A. Skolnick, JAMA262:1433 (1989); P. Nickerson et al., Ann. Intern. Med. 111:851-853(1989); Anonymous, Clinica 354:16 (1989); L. V. Kirchhoff, Ann. Intern.Med. 111:773-775 (1989); G. Bonfim et al., ISBT/AABB Joint Congress,Abstract S445, 112 (Nov. 10-15, 1990); P. Kerndt et al., Transfusion28:S108 (1988); I. H. Grant et al., Ann. Intern. Med. 111:849-851(1989); M. Boxaca et al., Int Conf. AIDS 6:437 (Abstract 3141) (1991);S. G. Sandler, Am. Red Cross Blood Services Letters 89:1-10 (1989); A.L. Bittencourt, Am. J. Dis. Child 130:97-103 (1976); R. Hoff et al.,Trans. R. Soc. Trop. Med. Hyg. 72:247-250 (1978); M. D. Gudino et al.,in Emerging Global Patterns in Transfusion-Transmitted Infections, R. G.Westphal et al., eds., Arlington, Va., American Association of BloodBanks, 65-86 (1990); I. G. Kagan et al., Rev. Biol. Trop. 14:55-73(1966); I. C. P. Dias et al., Mem. Inst. Oswaldo Cruz 79:139-147 (1984);and J. H. Maguire et al., "American Trypanosomiasis" in InfectiousDiseases, P. D. Hoepricke et al., eds, Philadelphia: J. P. Lippincott,pp. 1257-1266 (1989).

Diagnosis of the disease commonly is accomplished by identification ofparasites in the blood, cerebrospinal fluid, fixed tissue or lymph nodeduring periods of high fever; however, the organisms may be difficult todetect during the latent (or so-called indeterminant) phase, or duringchronic stages of infection. In xenodiagnosis, the intestinal contentsof insect vectors are examined for T. cruzi several weeks after theseparasites feed on the blood of a suspected patient. However, thisprocedure is laborious and lacks sensitivity. E. L. Segura,"Xenodiagnosis" in Chagas' Disease Vectors, R. R. Brenner et al., eds.,11:41-45, Boca Raton, Fla., CRC Press (1987)

Several different serologic methodologies have been used to diagnoseChagas' Disease. These methodologies include indirectimmunofluorescence, indirect hemagglutination, complement fixation andenzyme immunoassay. See, for example, F. Zicker et al., Bull. WorldHealth Organ. 68:465-471 (1990); M. E. Carmargo, Rev. Inst. Med. Trop.Sao Paulo 8:227-234 (1977); M. E. Carmargo et al., Bull. Pan Am. HealthOrgan 19:233-264 (1985); A. A. Pan et al., J. Infect. Dis. (165:585-588[1992]); A. A. Pan et al., Am. J. Trop. Med. Hyg. 45:120 Abstract 66(1991); A. F. Ferreira et al., Rev. Inst. Med. Trop. San Paulo33:123-128 (1991). Specific antibody responses to the parasite aredetectable after infection, and the titers typically remain high forlife. D. M. Israelski et al., Am. J. Trop. Med. Hyg. 39:445-455 (1988);R. Lelchuck et al., Clin. Exp. Immunol. 6:547-555 (1970); N. H. Vattuoneet al., Am. J. Trop. Med. Hyg. 76:45-47 (1973).

The natural transmission of Chagas' Disease in humans occurs when skinor mucosa come in contact with feces of infected bugs. The signs andsymptoms are so mild that the recent infection usually is not associatedwith T. cruzi infection. Even without treatment, the majority ofindividuals recover from the acute stages of disease. After a latentperiod of years or decades, a percentage of individuals (20-40%) developthe cardiac or gastrointestinal symptoms that characterize chronicChagas' Disease. Persistence of parasitemia in asymptomatic individualsand survival of the parasite in banked blood and blood componentsincreases the dangers of transmission of Chagas' Disease by bloodtransmision; infected blood used for transfusion in blood banks poses asignificant public health problem. A rapid and reliable assay withstandardized components for screening and confirming blood donors forChagas' Disease thus would be extremely useful in preventing transfusionof the disease by blood banks.

SUMMARY OF THE INVENTION

The present invention provides a method which can be utilized as aconfirmatory test for the presence of antibodies to T. cruzi in a testsample. The method comprises (a) determining the presence of a first T.cruzi antibody in a test sample, which comprises (i) contacting a firstaliquot of a test sample with a first T. cruzi antigen attached to asolid support to form a mixture and incubating same to form firstantigen/antibody complexes, (ii) contacting said first antigen/antibodycomplexes with an indicator reagent for a time and under conditionssufficient to form first antigen/antibody/indicator reagent complexes,and (iii) detecting the presence of the first T. cruzi antibody bymeasuring the signal generated; (b) determining the presence of a secondT. cruzi antibody in a test sample, which comprises (i) contacting asecond aliquot of a test sample with a second T. cruzi antigen attachedto a solid support to form a mixture and incubating same to form secondantigen/antibody complexes, (ii) contacting said second antigen/antibodycomplexes with an indicator reagent for a time and under conditionssufficient to form second antigen/antibody/indicator reagent complexes,and (iii) detecting the presence of the second T. cruzi antibody bymeasuring the signal generated; and (c) determining the presence of athird T. cruzi antibody in a test sample, which comprises (i) contactinga third aliquot of a test sample with a third T. cruzi antigen attachedto a solid support to form a mixture and incubating same to form thirdantigen/antibody complexes, (ii) contacting said third antigen/antibodycomplexes with an indicator reagent for a time and under conditionssufficient to form third antigen/antibody/indicator reagent complexesand (iii) detecting the presence of the third T. cruzi antibody bymeasuring the signal generated. The presence of at least two specificantibodies confirms the presence of T. cruzi antibody in the testsample. T. cruzi antigens for use in the assay include Gp90, Gp 60/50and LPPG. The antigens are used with the proviso that the antigen usedas the first antigen is not used for the second antigen in step (b) orthe third antigen in step (c), the antigen used as the second antigen isnot used for the first antigen in step (a) or the third antigen in step(c), and the antigen used as the third antigen is not used for the firstantigen in step (a) or the second antigen in step (b). The indicatorreagent used in the assay comprises a label which is selected from thegroup consisting of a chromogen, a catalyst, a luminescent compound, achemiluminescent compound, a radioactive element, and a direct visuallabel.

The present invention also provides diagnostic reagents for use in amethod for detection of T. cruzi antibody. These reagents comprise Gp90,Gp 60/50 and LPPG. Also provided are diagnostic test kits forconfirmatory tests which include Gp 90, Gp60/50 and LPPG.

In addition, processes for purifying the Gp60/50 and the LPPG antigensalso are provided. The process for purifying the Gp60/50 antigen of T.cruzi, comprises (a) isolating the membrane of the epimastigote stage ofT. cruzi by dounce homogenization; (b) extracting the Gp60/50 antigen;(c) applying the resultant extract of step (b) to a Galanthus nivalislectin affinity column and eluting with a carbohydrate; and (d)purifying the eluate with an affinity column comprising a monodonalantibody specific for Gp60/50. The extraction step preferably isperformed with a non-ionic detergent.

Further, the present invention provides a process for linking anantigenic glycolipid of T. cruzi to a protein carrier, which comprises(a) obtaining the glycolipid lipophosphonopeptidoglycan (LPPG) from theepimastogote stage of T. cruzi; (b) linking LPPG of step (a) with aprotein using ethyldimethyl-amino-propyl-carbodiimide (EDAC) by (i)contacting LPPG with EDAC to form a resultant mixture and incubatingsaid mixture for a time and under conditions sufficient to activate themixture, (ii) incubating the activated mixture with the protein for atime and under conditions sufficient to link the LPPG with the protein;(c) purifying the mixture; and (d) eluting the LPPG from the mixture.The protein preferred to be linked is bovine serum albumin (BSA).Further, it is preferred that the purification of step (c) is performedby passing the mixture over blue dextran Sepharose.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a bar graph wherein the number of sera from a negativepopulation reactive with T. cruzi. Gp90 (90 kD) is shown along thevertical axis while the Signal to Negative ratio (S/Neg) is shown alongthe horizontal axis of the graph, wherein the number of sera tested was289.

FIG. 1B is a bar graph wherein the number of sera from a negativepopulation reactive with T. cruzi Gp60/50 is shown along the verticalaxis while the Signal to Negative ratio (S/Neg) is shown along thehorizontal axis of the graph, wherein the number of sera tested was 289.

FIG. 1C is a bar graph wherein the number of sera from a negativepopulation reactive with T. cruzi LPPG is shown along the vertical axiswhile the Signal to Negative ratio (S/Neg) is shown along the horizontalaxis of the graph, wherein the number of sera tested was 289.

FIGS. 2A through 2D show the results of the assay of the inventiongraphed as a function of Optical Density (OD) versus the dilution factorof the (previously positive) tested sera, wherein a solid circleindicates gp90 bead, an open circle indicates gp60/50 bead, and an opentriangle indicates LPPG-BSA bead.

FIG. 2A is a graph of sample C, obtained from San Antonio, Tex.;

FIG. 2B is a graph of sample b, obtained from Los Angeles, Calif.;

FIG. 2C is a graph of a sample from Brazil;

FIG. D is a Chagas' Disease EIA Latin American Positive Control (AbbottLaboratories, Abbott Park, Ill.).

FIG. 3 is a scheme for confirmation of seropositivity for T. cruzi.

DETAILED DESCRIPTION OF THE INVENTION

An assay for the detection of T. cruzi antibody analyte in a test sampleis provided. The assay preferably is performed as an immunoassay,although the present invention is not limited to immunoreactive assays.Any assay utilizing specific binding members can be performed. A"specific binding member," as used herein, is a member of a specificbinding pair. That is, two different molecules where one of themolecules through chemical or physical means specifically binds to thesecond molecule. Therefore, in addition to antigen and antibody specificbinding pairs of common immunoassays, other specific binding pairs caninclude biotin and avidin, carbohydrates and lectins, complementarynucleotide sequences, effector and receptor molecules, cofactors andenzymes, enzyme inhibitors and enzymes, and the like. Furthermore,specific binding pairs can include members that are analogs of theoriginal specific binding member, for example, an analyte-analog.Immunoreactive specific binding members include antigens, antigenfragments; antibodies and antibody fragments, both monoclonal andpolyclonal; and complexes thereof, including those formed by recombinantDNA methods.

"Analyte," as used herein, is the substance to be detected which may bepresent in the test sample. The analyte can be any substance for whichthere exists a naturally occurring specific binding member (such as, anantibody), or for which a specific binding member can be prepared. Thus,an analyte is a substance that can bind to one or more specific bindingmembers in an assay. "Analyte" also includes any antigenic substances,haptens, antibodies, and combinations thereof. As a member of a specificbinding pair, the analyte can be detected by means of naturallyoccurring specific binding partners (pairs) such as the use of intrinsicfactor protein in the capture and/or indicator reagents for thedetermination of vitamin B₁₂, or the use of a lectin in the captureand/or indicator reagents for the determination of a carbohydrate. Theanalyte can include a protein, a peptide, an amino acid, a hormone, asteroid, a vitamin, a drug including those administered for therapeuticpurposes as well as those administered for illicit purposes, abacterium, a virus, and metabolites of or antibodies to any of the abovesubstances.

The test sample can be a mammalian biological fluid such as whole bloodor whole blood components including red blood cells, white blood cellsincluding lymphocyte or lymphocyte subset preparations, platelets, serumand plasma; ascites; saliva; stools; cerebrospinal fluid; urine; sputum;trachael aspirates and other constituents of the body which may containor be suspected of containing the analyte(s) of interest. The testsample also can be a culture fluid supernatant, or a suspension ofcultured cells. Mammals or others whose body fluids can be assayed forT. cruzi antibody analyte according to the present invention includehumans and primates, as well as other mammals who are suspected ofcontaining these analytes of interest.

The indicator reagent comprises a label conjugated to a specific bindingmember of each analyte. Each indicator reagent produces a detectablesignal at a level relative to the amount of the analyte, if any, presentin the test sample. In a preferred embodiment, each indicator reagent,while comprising a specific binding member of a different analyte, isconjugated to the same signal generating compound (label), which iscapable of generating a detectable signal. In general, the indicatorreagent is detected or measured after it is captured on the solid phasematerial. In the present invention, the total signal generated by theindicator reagent(s) indicates the presence of one or more of theanalytes in the test sample. It is contemplated that different signalgenerating compounds can be utilized in the practice of the presentinvention. Thus, for example, different fluorescent compounds could beutilized as the signal generating compounds, one for each indicatorreagent, and detection could be determined by reading at differentwavelengths. Or, a short-lived chemiluminescent compound such as anacridinium or phenanthridium compound and a long-lived chemiluminescentcompound such as a dioxetane can be utilized to generate signals atdifferent times for different analytes. Methods which detail the use oftwo or more chemiluminescent compounds which are capable of generatingsignals at different times are the subject matter of co-pending patentapplication U.S. Ser. No. 636,038, which enjoys common ownership and isincorporated herein by reference. Acridinium and phenanthridiniumcompounds are described in co-pending U.S. patent application Ser. No.07/271,763 filed Jun. 23, 1989, which enjoys common ownership and isincorporated herein by reference.

In addition to being either an antigen or an antibody member of aspecific binding pair, the specific binding member of the indicatorreagent can be a member of any specific binding pair, including eitherbiotin or avidin, a carbohydrate or a lectin, a complementary nucleotidesequence, an effector or a receptor molecule, an enzyme cofactor or anenzyme, an enzyme inhibitor or an enzyme, and the like. Animmunoreactive specific binding member can be an antibody, an antigen,or an antibody/antigen complex that is capable of binding either to theanalyte as in a sandwich assay, to the capture reagent as in acompetitive assay, or to the ancillary specific binding member as in anindirect assay. If an antibody is used, it can be a monoclonal antibody,a polyclonal antibody, an antibody fragment, a recombinant antibody, amixture thereof, or a mixture of an antibody and other specific bindingmembers. The details of the preparation of such antibodies and theirsuitability for use as specific binding members are well known to thosein the art.

The signal generating compound (label) of the indicator reagent iscapable of generating a measurable signal detectable by external means.The various signal generating compounds (labels) contemplated includechromagens; catalysts such as enzymes for example, horseradishperoxidase, alkaline phospatase, and B-galactosidase; luminescentcompounds such as fluorescein and rhodamine; chemiluminescent compoundssuch as acridinium compounds, phenanthridinium compounds and dioxetanecompounds; radioactive elements; and direct visual labels. The selectionof a particular label is not critical, but it will be capable ofproducing a signal either by itself or in conjunction with one or moreadditional substances. A variety of different indicator reagents can beformed by varying either the label or the specific binding member.

The capture reagents of the present invention comprise a specificbinding member for each of the analytes of interest which are attachedto at least one solid phase and which are unlabeled. Although thecapture reagent is specific for the analyte as in a sandwich assay, itcan be specific for an indicator reagent or analyte in a competitiveassay, or for an ancillary specific binding member, which itself isspecific for the analyte, as in an indirect assay. The capture reagentcan be directly or indirectly bound to a solid phase material before theperformance of the assay or during the performance of the assay, therebyenabling the separation of immobilized complexes from the test sample.This attachment can be achieved, for example, by coating the specificbinding member onto the solid phases by absorption or covalent coupling.Coating methods, and other known means of attachment, are known to thosein the art.

The specific binding member of the capture reagent can be any moleculecapable of specifically binding with another molecule. The specificbinding member of the capture reagent can be an immunoreactive compoundsuch as an antibody, antigen, or antibody/antigen complex. If anantibody is used, it can be a monodonal antibody, a polyclonal antibody,an antibody fragment, a recombinant antibody, a mixture thereof, or amixture of an antibody and other specific binding members.

The "solid phase" is not critical and can be selected by one skilled inthe art. Thus, latex particles, microparticles, magnetic or non-magneticbeads, membranes, plastic tubes, wails of wells of reaction trays, glassor silicon chips and tanned sheep red blood cells are all suitableexamples. Suitable methods for immobilizing capture reagents on solidphases include ionic, hydrophobic, covalent interactions and the like.

A "solid phase", as used herein, refers to any material which isinsoluble, or can be made insoluble by a subsequent reaction. The solidphase can be chosen for its intrinsic ability to attract and immobilizethe capture reagent. Alternatively, the solid phase can retain anadditional receptor which has the ability to attract and immobilize thecapture reagent. The additional receptor can include a charged substancethat is oppositely charged with respect to the capture reagent itself orto a charged substance conjugated to the capture reagent. As yet anotheralternative, the receptor molecule can be any specific binding memberwhich is immobilized upon (attached to) the solid phase and which hasthe ability to immobilize the capture reagent through a specific bindingreaction. The receptor molecule enables the indirect binding of thecapture reagent to a solid phase material before the performance of theassay or during the performance of the assay. The solid phase thus canbe a plastic, derivatized plastic, magnetic or non-magnetic metal, glassor silicon surface of a test tube, microtiter well, sheet, bead,microparticle, chip, and other configurations known to those of ordinaryskill in the art.

It is contemplated and within the scope of the invention that the solidphase also can comprise any suitable porous material with sufficientporosity to allow access by detection antibodies and a suitable surfaceaffinity to bind antigens. Microporous structures are generallypreferred, but materials with gel structure in the hydrated state may beused as well. Such useful solid supports include:

natural polymeric carbohydrates and their synthetically modified,cross-linked or substituted derivatives, such as agar, agarose,cross-linked alginic acid, substituted and cross-linked guar gums,cellulose esters, especially with nitric acid and carboxylic acids,mixed cellulose esters, and cellulose ethers; natural polymerscontaining nitrogen, such as proteins and derivatives, includingcross-linked or modified gelatins; natural hydrocarbon polymers, such aslatex and rubber; synthetic polymers which may be prepared with suitablyporous structures, such as vinyl polymers, including polyethylene,polypropylene, polystyrene, polyvinylchloride, polyvinylacetate and itspartially hydrolyzed derivatives, polyacrylamides, polymethacrylates,copolymers and terpolymers of the above polycondensates, such aspolyesters, polyamides, and other polymers, such as polyurethanes orpolyepoxides; porous inorganic materials such as sulfates or carbonatesof alkaline earth metals and magnesium, including barium sulfate,calcium sulfate, calcium carbonate, silicates of alkali and alkalineearth metals, aluminum and magnesium; and aluminum or silicon oxides orhydrates, such as clays, alumina, talc, kaolin, zeolite, silica gel, orglass (these materials may be used as filters with the above polymericmaterials); and mixtures or copolymers of the above classes, such asgraft copolymers obtained by initializing polymerization of syntheticpolymers on a pre-exisitng natural polymer. All of these materials maybe used in suitable shapes, such as films, sheets, or plates, or theymay be coated onto or bonded or laminated to appropriate inert carriers,such as paper, glass, plastic films, or fabrics.

The porous structure of nitrocellulose has excellent absorption andadsorption qualities for a wide variety of reagents including monoclonalantibodies. Nylon also possesses similar characteristics and also issuitable.

It is contemplated that such porous solid supports described hereinaboveare preferably in the form of sheets of thickness from about 0.01 to 0.5mm, preferably about 0.1 mm. The pore size may vary within wide limits,and is preferably from about 0.025 to 15 microns, especially from about0.15 to 15 microns. The surfaces of such supports may be activated bychemical processes which cause covalent linkage bf the antigen orantibody to the support. The irreversible binding of the antigen orantibody is obtained, however, in general, by adsorption on the porousmaterial by poorly understood hydrophobic forces.

Preferred solid phase materials for flow-through assay devices includefilter paper such as a porous fiberglass material or other fiber matrixmaterials. The thickness of such material is not critical and will be amatter of choice, largely based upon the properties of the sample oranalyte being assayed, such as the fluidity of the test sample.

To change or enhance the intrinsic charge of the solid phase, a chargedsubstance can be coated directly to the material or onto microparticleswhich then are retained by a solid phase support material.Alternatively, microparticles can serve as the solid phase, by beingretained in a column or being suspended in the mixture of solublereagents and test sample, or the particles themselves can be retainedand immobilized by a solid phase support material. By "retained andimmobilized" is meant that the particles on or in the support materialare not capable of substantial movement to positions elsewhere withinthe support material. The particles can be selected by one skilled inthe art from any suitable type of particulate material and include thosecomposed of polystyrene, polymethylacrylate, polypropylene, latex,polytetrafluoroethylene, polyacrylonitrile, polycarbonate, or similarmaterials. The size of the particles is not critical, although it ispreferred that the average diameter of the particles be smaller than theaverage pore size of the support material being used. Thus, embodimentswhich utilize various other solid phases also are contemplated and arewithin the scope of this invention. For example, ion capture proceduresfor immobilizing an immobilizable reaction complex with a negativelycharged polymer, described in co-pending U.S. patent application Ser.No. 150,278 corresponding to EP Publication No. 0326100, and U.S. patentapplication Ser. No. 375,029 (EP Publication No. 0406473), which enjoycommon ownership and are incorporated herein by reference, can beemployed according to the present invention to effect a fastsolution-phase immunochemical reaction. An immobilizable immune complexis separated from the rest of the reaction mixture by ionic interactionsbetween the negatively charged polyanion/immune complex and thepreviously treated, positively charged porous matrix and detected byusing various signal generating systems previously described, includingthose described in chemiluminescent signal measurements as described inco-pending U.S. patent application Ser. No. 921,979 corresponding to EPOPublication No. 0 273,115, which enjoys common ownership and which isincorporated herein by reference.

Also, the methods of the present invention can be adapted for use insystems which utilize microparticle technology including automated andsemi-automated systems wherein the solid phase comprises amicroparticle. Such systems include those described in pending U.S.patent application Ser. No. 425,651 and U.S. Pat. No. 5,089,424, whichcorrespond to published EPO applications Nos. EP 0 425 633 and EP 0 424634, respectively, and U.S. Pat. No. 5,006,309 all of which enjoy commonownership and are incorporated herein by reference. Such systems alsoinclude U.S. patent application Ser. No. 07/859,218 filed Mar. 27, 1992,which enjoys common ownership and is incorporated herein by reference.

One aspect of the invention is a confirmatory assay for antibody to T.cruzi, the agent of Chagas' Disease. This assay involves coating a firstsolid phase with a first T. cruzi antigen, coating a second solid phasewith a second T. cruzi antigen, and coating a third solid phase with athird T. cruzi antigen. An aliquot of a test sample, previously screenedfor T. cruzi antibody and repeat reactive in a screening test, is placedin contact with each solid phase. Thus, an aliquot of a test sample isseparately placed in contact with each solid phase and reactedseparately. The T. cruzi antigen(s) preferred for use in thisconfirmatory assay are Gp90, Gp 60/50 and LPPG-BSA. The resultantmixture is incubated for a time and under conditions sufficient to formantigen-antibody complexes. Then, an indicator reagent specific for eachantibody and attached to a label capable of generating a measurablesignal is contacted with the antigen-antibody complexes and incubatedfor a time and under conditions sufficient to formantigen/antibody/indicator reagent. The signal generated from each solidphase is determined. The presence of T. cruzi is determined as afunction of the signal generated from the solid phase-indicator reagent.The presence of antibody to at least two of the three T. cruzi antigensindicates the confirmed presence of T. cruzi antibody in the testsample.

In a second aspect of the present invention, at least one antigen of thethree antigens disclosed above is coated on a solid support. A testsample is contacted with the solid phase and the resultant mixture isincubated for a time and under conditions sufficient to formantigen/antibody complexes. Then, an indicator reagent, whichspecifically reacts with the specific binding member(s) now attached tothe solid support and is capable of generating a measurable signal, isadded to the complexes, and the resultant second mixture is incubatedfor a time and under conditions sufficient to formantigen/antibody/indicator reagent. The signal generated from the solidphase/indicator reagent is measured; a signal generated which is greaterthan a pre-determined known negative control cut-off value is consideredreactive, and thus, indicative of the presence of antibody to T. cruzi.

The eukaryotic organism T. cruzi has greater than 30,000 proteins; thereis conversation of epitopes between members of the family ofTrypanosomidae/Kinetoplastida. D. E. Lanar et al., Mol. Biochem.Parasitol. 3:327-341 (1981) and S. P. Craig et al., Comp. Biochem.Physiol. 95B:667-662 (1990). Further diversity of T.cruzi includeszymodemes or strain variability from one geographic location to thenext. Xenodiagnosis must be performed with care to rule out chance ofcross-reaction with T. rangeli, a parasite which has similar insectvectors but different locations of metacyclic trypomastigotes. Theantigens used for the above described assay formats are purified tohomogeneity from either the amastigote or epimastigote stages of T.cruzi and subsequently attached (coated) onto a solid support. The Gp90and LPPG antigens previously have been characterized and the Gp90antigen (s) has been shown to be immunogenic. See, for example, M.Schechter et al., Lancet 2:939-941 (1983); L. V. Kirchhoff et al., J.Inf. Dis. 155:561-564 (1987) and J. O. Previato et al., J. Biol. Chem.265:2518-2526 (1990). However, the use of the combination of the threepreferred T. cruzi antigens has not been described heretofore for use ina confirmatory or screening assay for antibody to T. cruzi. In theconfirmatory assay, if the absorbance of a suspected sample is above thecutoff value in three-of-three or two-of-three determinations using T.cruzi antigens, it is considered as a confirmed positive sample for thedisease. However, if the absorbance is not above the cutoff in any or inonly one of three antigens utilized, the test sample is subjected toradioimmunoprecipitation RIPA. In RIPA, diagnostic bands exhibitcharacteristic banding patterns at 32, 34 and 90 kD. The titer of apositive test sample is reflected in 19 and 25 kD banding pattern. Thus,the test sample can fall into three categories: confirmed reactive,indeterminate reactive or negative.

The present invention will now be described by way of examples, whichare meant to illustrate, but not to limit, the spirit and scope of thepresent invention.

EXAMPLES Example 1 Culture of Parasites

Stock cultures of T. cruzi epimastigotes (available from the AmericanType Culture Collection, Rockville, Md.) were maintained in modified NNNmedium (disclosed in C. Pan, Am. J. Trop. Med. Hyg. 17:823-832) 1968)with an overlay of UM-55 medium (as disclosed in P. M. Rainey et al.,Mol. Biochem. Parasitol. 49:111-118 (1991); A. Pan, Exp. Parasitol.58:72-80 (1984) at 24° C. following the procedures described in S. C.Pan, Bull. World Health Organ. 60:101-107 (1982); P. M. Rainey et al.,Mol. Biochem. Parasitol. 41:111-118 (1991); and A. A. Pan et al., J.Immunol, 143:1001-1008 (1989). For experiments, epimastigotes wereinoculated into 5 ml of UM-55 medium and passaged once. Five millilitersof this culture in mid-log phase of growth was used to inoculate aspinner flask containing 6 liters (1) of UM-55 medium and then wasincubated for seven to ten days at 26° C.

Example 2 Production of T. cruzi Membranes

Epimastigotes or amastigotes of T. cruzi were grown to log phase,harvested and washed three times with phosphate buffered saline (PBS)(pH 7.4) by centrifugation at 6000×g. The final pellet of cells wasresuspended in lysis buffer (consisting of 20 mM Tris-HCl [pH 7.3], 40mM NaCl, 10 mM EDTA, 2 mM phenylmethyl sulfonyl fluoride ([PMSF] and 1mM iodoacetamide). The organisms (50 ml packed epimastigotes or 20 mlpacked amastigotes) were disrupted either by nitrogen cavitation (1500psi for 15 minutes on ice) or by dounce homogenization. The lysates weresubfractioned by differential centrifugation at 48,600×g for 30 minutesat 4° C., following the method described by A. A. Pan et al., supra.

Example 3 Preparation of T. cruzi Antigens for Assay Use

Gp 60/50. The Gp 60/50 glycoprotein of T. cruzi was purified fromepimastigotes as follows. Briefly, the membrane-enriched pellet wassolubilized for two hours at 4° C. in Buffer A (consisting of 20 mMTris-HCl, [pH 7.2]) containing 150 mM NaCl, 2.0 mM iodoacetamide, 1 mMEDTA, 0.5 mM PMSF, 77 μM Aprotonin and two percent (2%) NP-40, andcleared by centrifugation at 48,000×g for 30 minutes at 4° C. Theresultant supernatant fluid was applied to a 20 ml Gananthus nivulus(GNA) lectin column (available from E. Y. Laboratories, San Mateo,Calif.), equilibrated in Buffer A which contained 0.5M NaCl, 0.1% NP-40and 1 mM EDTA. The column was eluted with 0.3M alpha-methyl pyranosidein the same buffer. This eluate was applied to a monoclonal anti-Gp60/50 (A. A. Pan et al., supra) IgG Sepharose column inphosphate-buffered saline (PBS, pH 7.2), washed with PBS, and elutedwith 50 mM diethylamine as described in M. A. Winkler et al., Proc.Natl. Acad. Sci. 81:3054-3058 (1984). The antigen was analyzed by tenpercent (10%) SDS-polyacrylamide gel electrophoresis (SDS-PAGE) underreducing conditions (U. K. Laemmli, Nature 227:680-685 [1970]) andsubjected to silver-staining (available from Bio-Rad, Richmond, Calif.),as described in M. A. Winkler et al., supra. This novel process produceda homogeneous glycoprotein (at least 20% carbohydrate). The Gananthusnivulus column isolated the glycoprotein in 1-2% yield from the majorityof membrane-extracted proteins.

Gp 90. The Gp 90 kD antigen was isolated from axenically grownamastigote membranes using a similar technique as for GP 60/50, with thefollowing modifications. A lentil lectin Sepharose 4B column (availablefrom Pharmacia-LKB, Piscataway, N.J.) was substituted for the GNA lectincolumn, and the Buffer A used in the application, washing, and elutionof the column contained 50 mM Tris-HCl (pH 7.5), 0.5 mM CaCl₂, 0.5 mMMnCl₂, 0.5M NaCl and 0.1% NP-40. The eluted material was affinitypurified by a monoclonal anti-Gp 90 kD IgG2b(A. A. Pan et al., supra)Sepharose 4B column as described above. Protein concentration wasmeasured for the membranes and purified antigens by the Pierce CoomassieBlue G-250 dye binding assay (available from Pierce, Rockford, Ill.).This antigen also was analyzed by SDS-PAGE, but stained by CoomassieBrilliant Blue R-250. The novel process produced a homogeneousglycoprotein for use in capturing and detecting T. cruzi antibodies. Itwas determined that about 24 ng gave a high signal in assays. Also, theyield of glycoprotein was at least 300 μg per 18 l of Amastigotes.

Example 4 Preparation of Lipophosphonopeptidoglycan (LPPG) Antigen forAssay Use

LLPG was isolated from the whole epimastigotes by the procedure of J. O.Previato et al., J. Biol. Chem. 265:2518-2526 (1990). Briefly,epimastigotes were thawed, washed three times in water, and extractedwith 45 percent phenol in water. The aqueous phase was lyopkilized,applied to a P-100 gel filtration column in water, and the excluded peaklyophilized. The powder was extracted with chloroform:methanol:water(10:3:1), dried, then dissolved in water and precipitated with five (5)volumes of methanol at -20° C. LPPG was quantitated by orcinal-surfuricadd assay for carbohydrates, following the method of C. A. White et al.,"Oligosaccharides" in Carbohydrate analysis. A Practical Approach, M. F.Chaplin et al., eds. Washington D.C.: IRL Press, pp. 37-54 (1987). Thisglycolipid coated poorly onto the solid supports used (polystyrenebeads) and therefore, a process was developed for linking the LPPGantigenic glycolipid from T. cruzi to a protein carrier so that theconjugate could be purified by affinity chromatography on blue dextranand subsequently coated onto solid phases for diagnostic assay use. LPPGwas linked to bovine serum albumin (BSA) with ethyldiaminoprepylcarbodiimide (EDAC, available from Sigma Chem. Co., St.Louis, Mo.), following the methods disclosed in S. Bauminger et al.,Methods Enzymol. 70:151-159 (1980) and modified as follows. LPPG waslinked to bovine serum albumin (BSA) with EDAC in a two-step procedure,using a 1:2 mass ratio of LPPG to BSA. In the first step, LFPG wasreacted at room temperature with EDAC, and then the activated mixturewas reacted overnight with BSA. This material was purified by applyingthe mixture to a Blue dextran Sepharose (available from Sigma Chem. Co.,St. Louis, Mo.) affinity column (10×1 cm), washing in PBS and elutingthe LPPG-BSA conjugate with: 0.5M potassium thiocyanate. Titration ofantigen coating (100 ng to 20 μg per bead) showed that 30 ng per 1/4"bead was preferred. The resultant eluate can be diluted and directlycoated onto beads.

Example 5 Three Bead Confirmatory Enzyme Immunoassay Bead CoatingProcedure

Each of the above antigens were separately coated onto polystyrene beads(0.625 cm in size), as follows: Polystyrene beads (2000 beads, 0.635 cm,available from Abbott Laboratories, Abbott Park, Ill.) were washed withisopropanol-water (71:400) for 20 minutes at 22° C. The fluid wasremoved by aspiration. 400 ml of PBS containing antigen (either 60 μg ofLPPG-BSA prepared as in Example 4 or 60 μg of Gp60/50 prepared as inExample 3, or 48 μg of Gp90 prepared as in Example 3) was added to thebeads. The beads and antigens were contacted with agitation for 2 hoursat 40° C. Following agitation, the fluid was removed, and 400 ml of ablocking solution (3% bovine serum albumin in PBS) was added. Theresultant mixture was agitated for 1 hour at 40° C. Then the fluid wasremoved, and 400 ml of an over-coat solution (5% sucrose and 0.5%gelatin in water) was added, the resultant mixture was incubated withagitation for 20 minutes at 22° C. The fluid was drained, and the beadswere dried at 22° C. with nitrogen gas. The coated beads were storeddessicated at 4° C.

Immunoassay

Enzyme Immunoassay was performed as follows. 5 μl of a serum sample wasdiluted with 200 μl of specimen diluent in three separate reaction wellsof a tray, and then incubated with each of three antigen coated beads(prepared as described hereinabove) for one (1) hour at 40° C. The beadsthen were washed with distilled water (Abbott Quikwash®, available fromAbbott Laboratories, Abbott Park, Ill.), and incubated with conjugate(goat anti-human IgG [heavy and light chain] conjugated to horseradishperoxidase [available from Kierkegaard & Perry, Gaithersburg, Md.]) forthirty (30) minutes at 40° C. The beads then were washed, transferred totubes and incubated with 300 μl of OPD tablet (Abbott Laboratories,Abbott Park, Ill.) dissolved in OPD diluent (comprising 50 mMcitrate-phosphate containing 0.02% H₂ O₂) for thirty (30) minutes atroom temperature. The reaction was stopped by adding 1 ml of 1N H₂ SO₄,and the absorbance was analyzed using an Abbott Quantum®spectrophotometer at 492 nm. In all experiments, a negative control(recalcified human plasma) and a positive control (inactivated humanplasma, positive for antibody to T. cruzi) were included and assayed intriplicate. The cutoff value was determined from inspection of thesample absorbances in a negative population (SE Wisconsin; N=289). Thecutoff value (S/N) was 3.3 for LPPG; 3.5 for Gp60/50; and 3.5 for Gp 90.The presence or absence of antibody to T. cruzi in an unknown sample wasdetermined by relating the S/N to the cutoff value.

Example 6 Radioimmunoprecipitation (RIPA)

The epimastigote membrane enriched fraction obtained from Gp 60/50purification was solubilized in 10 mM Tris-HCl (pH 7.8), 150 mM NaCl, 1mM EDTA, 1 mM PMSF, 1 mM iodoacetamide, and 2.0% NP-40. The resultantmixture was equilibrated for one hour at 4° C., and the particulatematerial was separated from the soluble membrane protein bycentrifugation at 20,000×g for thirty (30) minutes at 4° C. Theresultant material was radiolabeled with Na ¹²⁵ I by chloramine-T methodas described by W. M. Hunter et al., Nature 194:495-496 (1962). Thelabeled antigen was pre-absorbed with normal human serum-coated proteinA Sepharose Cl-4B for one (1) hour at 4° C., and then it was centrifugedat 1000×g for ten (10) minutes. The unbound material (at 10⁷ cpm in atotal volume of 20 to 25 μl) was incubated with 10 μl of the test serumovernight on ice. A 50 μl aliquot of 50% protein-A Sepharose CL-4B(Sigma Chemical Co., St. Louis, Mo.) suspension in PBS was added to themixture and vortexed for 30 minutes at 4° C. The sample then was washedthree (3) times in PBS containing 1% NP-40 and once in PBS containing 1%NP-40 and 0.05% sodium dodecyl sulfate (SDS). The sample was boiled for5 minutes in SDS-loading buffer (2.3% SDS, 10% glycerol, 62.5 mMTris-HCl, pH 6.8), centrifuged (12,500×g for 5 minutes) then thesupernatant fluid was removed for analysis. TheSDS-polyacrylamide-gel-electrophoresis was carried out according to theprocedure of Laemmli (Nature 227:680-685 [1970]) in a 12.5%polyacrylamide gel. Autoradiography was conducted for 7-10 days at -70°C. with X-ray film (Kodak XAR-5) and Lightnight Plus intensifying screen(E. I. DuPont de Nemours & Co., Wilmington, Del.).

Example 7 Comparative Testing

Samples

Xenodiagnosis-positive samples were obtained from Institute FatalaChaben, Buenos Aires, Argentina. Malaria sera were obtained from Africa(the Gambia) and India (Madras, India). African leishmaniasis sera wereobtained from Sudan; schistosomiasis sera were obtained from Brazil.Negative samples, obtained from a "low risk" area of the United States(Milwaukee, Wis.), systemic lupus erythematosus (SLE) (Milwaukee, Wis.)and syphilis sera (Detroit, Mich.) also were assayed with theconfirmatory assay of the invention.

Dilution Panel of Positive Serum

Several sera positive for antibody to T. cruzi, as determined by theconfirmatory assay of the invention set forth in Example 5, were dilutedappropriately to produce a dilution panel for determination ofsensitivity. The positive control (human plasma heat inactivated at 56°C.) was diluted into normal human plasma to give an absorbance at 492 nmof 0.500 to 1.999, and was further diluted to 1:22 and 1:100. Severalother positive samples were similarly diluted to 1:4 (high positive);1:6 (medium positive); 1:9 (borderline positive); 1:13.5 (low positive);and 1:22 (non-reactive positive). Positive and negative controls alsowere tested (Abbott Laboratories, Abbott Park, Ill.). All sera wereanalyzed by the confirmatory assay of the invention and RIPA asdescribed above.

Evaluation

A study was performed to evaluate the confirmatory assay of theinvention and R/PA. Samples previously tested with a screening assay(Chagas Antibody EIA for Chagas' Disease, Abbott Laboratories, AbbottPark, Ill.) were used in the evaluation. These samples were obtainedfrom eight field sites selected from the southwestern United States; thesamples were unlinked except for classification at each specificlocation into Hispanic or non-Hispanic surname groups. Specimens wereidentified only by number for testing, the assays were performed, andthe assay results were decoded at a later date. Total number of samplesscreened was 13,109. These samples were obtained from the followingsites (Hispanic/non-Hispanic): Alburquerque, NM (224/224); Houston, Tex.(986/1326); McAllen, Tex. (664/259); San Antonio, Tex. (2396/1599); LosAngeles, Calif. (1050/1050); Sacramento, Calif. (1899/600); and SanDiego (416/416). Samples were S/N greater than 3.0 (N=112) using thescreening assay described above and were evaluated by the confirmatoryassay of the invention by following the procedure detailed in Example 5and RIPA according to the procedure of Example 6.

Results

1. Accuracy. The ability of the confirmatory assay of the invention todetect antibody to T. cruzi was established by comparison to consensuspositive samples (samples in which there were positive results by bothhemagglutination and indirect immunofluorescent antibody assays forChagas' Disease). The assay of the invention was utilized on these 82consensus positive samples. Fifty-six (56) samples demonstrated anabsorbance value above the cutoff in three of three beads (sensitivityof 68.3% [24/82]). Twenty-four (24) samples were above the cutoff inonly two of three antigens (29.3%) Two samples were positive on only oneof three antigens; these had to be confirmed by RIPA. As seen in Table1, the overall performance of the three-bead confirmatory assay was97.56% (80/82) on consensus positive samples.

                  TABLE 1                                                         ______________________________________                                                             Assay of  Assay of                                                            the       the     Sensi-                                 Sample      Sample   Invention-                                                                              Invention-                                                                            tivity                                 Type        Number   Positive  Negative                                                                              (%)*                                   ______________________________________                                        Xenodiagnosed                                                                             28       28        0       100                                    Positive                                                                      Consensus   82       80        2       97.56                                  Positive**                                                                    Lesihmaniasis                                                                             7        0         7                                              Malaria     6        0         6                                              Toxoplasmosis                                                                             6        0         6                                              Schistosomiasis                                                                           10       0         10                                             Leprosy     1        0         1                                              Syphilis    2        0         2                                              Systemic lupus                                                                            5        0         5                                              High Rheumatoid                                                                           2        0         2                                              Factor                                                                        ______________________________________                                         *Sensitivity (%) = (No. ChagasEIA Positive/No. Xenodiagnosis Positive)        × 100;                                                                  **Consensus Positive are reactive by Hemagglutination and                     Immunofluorescence assays                                                

Two hundred eighty-fine negative samples from a "low risk" region(southeast Wisconsin) also were assayed by the confirmatory EIA. As seenin FIG. 1A, FIG. 1B and FIG. 1C, the two hundred eighty-four sampleswere below the cutoffs in at least two of the three kinds ofantigen-coated beads. Five samples demonstrated an absorbance value(S/N) above the cutoff in one of three beads. However, sinceconfirmation is determined by reaction above the cutoff values in atleast two of three beads, the overall performance of the confirmatoryassay of the invention was 100% (289/289).

Confirmatory EIA of Xenodiagnosis-Positive Samples

A sample positive by xenodiagnosis represented a sample in which therewas a greater degree of certainty as to an individual having Chagas'Disease and thus, antibody to T. cruzi. Table 1 summarizes the data onthe confirmatory assay of the invention on these samples. As the datafrom the Table 1 demonstrate, all samples showed an absorbance above thecutoff value with three-of-three or two-of-three beads. No samples hadto be retested by RIPA. The overall agreement on the confirmatory assaywas 100% (28/28) on xenodiagnosis-positive samples.

Evaluation of the Three-Bead Confirmatory Assay with a Dilution Panel

The assay of the invention was tested with four consensus positivesamples diluted two- and three-fold from 1:2 to 1:96. The results ofthese tests are shown in FIG. 2A, FIG. 2B, FIG. 2C and FIG. 2D. As canbe seen in THIS FIGURE, all of the sera tested were positive (above thecutoff value in at least two of three antigen beads) by the confirmatoryassay of the invention through a dilution of 1:16. The samples wereconsidered consensus positive when tested by the Chagas US Screen EIA(Abbott Laboratories, Abbott Park, Ill.), hemagglutination and indirectimmunofluorescent antibody. All samples were diluted two- or three-foldto a dilution of 1:96.

Cross-Reactivity

Specimens from patients with other parasitic diseases were tested withthe confirmatory assay of the invention. As the data in Table 1demonstrate, absorbances were below the cutoff value, which indicated nocross-reactivity in the assay.

Evaluation of Three-Bead Confirmatory Assay with Samples from the U.S.Prevalence Study

In an in-house evaluation, 13,109 blood samples donated in southwesternareas of the U.S. were screened with a Abbott Chagas Antibody EIA(Abbott Laboratories, Abbott Park, Ill.). All samples withsignal-to-negative (S/N) greater than 3.0 were assayed by theconfirmatory assay of the invention as detailed in Example 5.Thirty-four (34) samples in the screening assay were "RR" (repeatreactive). As seen in Table 2, nine (9) samples were positive in atleast two-of-three beads, two samples were positive in one-of-threebeads, and two samples were negative on all three beads. Four samples(two negative on all three beads and two positives on only one bead)were indeterminate and required additional analysis by RIPA.

                  TABLE 2                                                         ______________________________________                                        Confirmatory Assay of the Invention Tested with Positive Sera                             Positive Sample Type                                                                              In-House                                                  Xenodiagnosis                                                                           Consensus*                                                                              Study                                         ______________________________________                                        Confirmatory Assay                                                            Results                                                                       3/3 Positive  21          56        7                                         2/3 Positive  7           24        2                                         1/3 Positive  0            2         3+                                       0/3 Positive  0            0         2+                                       ______________________________________                                         *Samples in which Hemagglutination and Immunofluorescence Assays were         positive in both tests                                                        +Samples to be confirmed by RIPA                                         

RIPA FIG. 3 represents the results of a RIPA of 125I-labeled solubilizedmembrane extract of epimastigotes of T. cruzi with the dilution panel ofpositive sera for antibody to T. cruzi. Nine (9) major proteins wererevealed under reducing conditions; these bands had molecular weights(Mr) of 19, 25, 28, 32, 34, 44, 58, 69 and 90 kD (kilodaltons). Thereappeared to be two (2) bands of Mr 32 and 34 kD which were stronglyprecipitated, while the 90 kD band was of medium intensity; these threebands appeared to be the most diagnostic proteins. The 19 and 25 kDbands appeared to be dependent on the titer of serum, being present inthe positive control 1:22, 1:100, 1:4 (high positive); and the screeningtest's positive control (previously described above). The 19 and 25 kDbands were absent in the 1:6 (mediium positive); 1:9 (borderlinepositive); 1:13.5 (low positive); 1:22 (non-reactive positive); and thenegative control for the Chagas' Disease screening assay previouslydescribed herein. The bands which immunoprecipitated at 28, 44, 58 and69 kD appeared in various negative samples tested and were non-specific.As demonstrated, the 32, 34 and 90 kD bands appeared in all of thedilutions tested.

Comparison of RIPA to Xenodiagnosis-Positive Samples

All 28 xenodiagnosis-positive samples were analyzed by RIPA. All samplesshowed the characteristic diagnostic bands at 32, 34 and 90 kD. Severalof these sera also demonstrated high titer bands at 19 and 25 kD. Theoverall agreement on the RIPA was 100% (28/28) on xenodiagnosis-positivesamples.

RIPA of Samples from U.S. Prevalence Study

To demonstrate the utility of RIPA, 122 samples from the U.S. prevalencestudy described above with signal/negative (S/N) greater than 3.0 wereassayed as follows. Samples with S/N from 3.0 to 5.0 (N=100) werenegative in RIPA. As Table 3 below demonstrates, of the 22 sampleshaving S/N greater than 5.0, 13 samples were confirmed positive. Four ofthe five indeterminate samples from the confirmatory assay confirmedpositive, and one sample remained indeterminate. Seven (7) samplesdemonstrated bands at 19 and 25 kD, which indicated high titer sera.

                  TABLE 3                                                         ______________________________________                                        Comparison of the 3-Bead Assay Versus RIPA                                              3-BEAD ASSAY RESULT                                                                             RIPA                                              Sample Type 3 of 3  2 of 3  1 of 3                                                                              0 of 3                                                                              Positive                              ______________________________________                                        Xenodiagnosed                                                                             21      7       0     0     28                                    Positives                                                                     Consensus   56      24      2     0     82                                    Positives                                                                     In-House US  7      2       3     2     13                                    Field Study                                                                   ______________________________________                                    

As shown by the data presented above, both the confirmatory assay of thepresent invention and RIPA had a clinical sensitivity of 100% whentested against xenodiagnosis-positive sera. When tested with consensuspositive sera, the confirmatory assay of the invention had a sensitivityof 97.56% (80/82). The remaining 2.4% (2/82) showed reactivity with oneof the three beads. When assayed against negative samples from a "lowrisk" region of southeast Wisconsin, the confirmatory assay of theinvention had a specificity of >99.99%. The proposed scheme forconfirmation is shown in FIG. 3. Thus, it can be seen that test samplesreactive with two-of-three or three-of-three T. cruzi antigens isconsidered a confirmed reactive seropositive sample. If the absorbanceof a sample is only above the cutoff value in one of the three antigens,or the sample is not above a cutoff with all three antigens, the sampleis considered as negative, or it is subjected to RIPA. In RIPA, testsamples are confirmed reactive when three-of-three or two-of-threediagnostic bands precipitate. Samples immunoprecipitating only one bandare considered indeterminant, and samples which do not immunoprecipitateany bands are considered negative. The confirmatory assay of theinvention provides a means of reducing the number of samples which needto be assayed by RIPA. This is beneficial because RIPA is considered avery time-consuming and labor-intensive procedure when compared to theassay of the present invention: the confirmatory assay of the inventionrequires approximately two hours to perform, while a RIPA may takeupwards of ten days for a result. RIPA previously has been used toconfirm the presence of T. cruzi antibody, wherein a protein extract ofsurface radiolabeled parasites was used when testing pedigreed reactivesamples. Protein bands at 72 and 90 kD in RIPA appeared to be sensitiveand specific. These antigens appeared to be highly conserved across awide geographical selection of strains of the parasite. However, RIPAhas practical limitations in that radioisotopes with short half-life areused, the technique is time-consuming, and the technique is not readilyadaptable to large scale screening. R. C. K. Wong et al., Trans. R. Soc.Trop. Med. Hyg. 80:275-281 (1986).

It is contemplated that the assay of the invention can be optimized evenfurther by varying assay conditions and/or incubation times, usingvarious combinations of antigen or antibody capture or probe reagents,and other methods, reagents and conditions known to those skilled in theart. The variance of the antibody capture reagent may then require theuse of a different antigen capture reagent. All these variations arecontemplated to be within the scope of this invention. Also, while someof the assays described in the examples used an automated system, it iswell within the scope of the present invention that manual methods orother automated analyzers can be used or adapted to the assay of thepresent invention. Therefore, the present invention is meant to belimited only by the appended claims.

What is claimed is:
 1. A Gp60/50 antigen made by a process comprising:a.isolating the membrane of the epimastigote stage of T. cruzi by douncehomogenization; b. extracting the Gp60/50 antigen; c. applying theresultant extract of step (b) to a Galanthus nivalis lectin affinitycolumn and eluting with a carbohydrate; d. purifying the eluate with anaffinity column comprising a monoclonal antibody specific for Gp60/50.